Molecular epidemiology of hospital-onset methicillin-resistant Staphylococcus aureus infections in Southern Chile.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of public health importance. In Chile, the Cordobes/Chilean clone was the predominant healthcare-associated MRSA (HA-MRSA) clone in 1998. Since then, the molecular epidemiological surveillance of MRSA has not been performed in Southern Chile. We aimed to investigate the molecular epidemiology of HA-MRSA infections in Southern Chile to identify the MRSA clones involved, and their evolutionary relationships with epidemic international MRSA lineages. A total of 303 single inpatient isolates of S. aureus were collected in the Valdivia County Hospital (2007-2008), revealing 33% (100 MRSA/303) prevalence for HA-MRSA infections. The SCCmec types I and IV were identified in 97% and 3% of HA-MRSA, respectively. All isolates lacked the pvl genes. A random sample (n = 29) of all MRSA was studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), SCCmec subtyping, agr and spa typing, and virulence genes profiling. PFGE analysis revealed the predominance (89%, 26/29) of pulsotype A and three additional pulsotypes, designated H1, I33, and G1. Pulsotype A (ST5-SCCmecI-spa-t149) is clonally related to the Cordobes/Chilean clone. Pulsotype H1 (ST5-SCCmecIVNT-spa-t002) is genetically related to the Pediatric clone (ST5-SCCmecIV). Pulsotype I33 (ST5-SCCmecIVc-spa-t002) is clonally related by PFGE to the community-associated MRSA (CA-MRSA) clone spread in Argentina, I-ST5-IVa-PVL(+). The G1 pulsotype (ST8-SCCmecIVc-spa-t024) is clonally related to the epidemic USA300 CA-MRSA. Here, we demonstrate the stability of the Cordobes/Chilean clone over time as the major HA-MRSA clone in Southern Chile. The identification of two CA-MRSA clones might suggest that these clones have entered into the healthcare setting from the community. These results emphasize the importance of the local surveillance of MRSA infections in the community and hospital settings.

RXRalpha regulates the pregnancy-specific glycoprotein 5 gene transcription through a functional retinoic acid responsive element.

Human pregnancy-specific glycoproteins (PSG) are major placental polypeptides encoded by eleven highly conserved genes expressed by the syncytiotrophoblast. The minimal promoter region of all PSG genes contains a putative Retinoic Acid Responsive Element (RARE) though the ability of retinoids to regulate PSG gene expression has not been established. Retinoid signaling pathway plays a key role for overall placenta biology and is essential for trophoblast differentiation. In this work, we investigated the participation of the RARE motif in the regulation of PSG5 gene transcription by retinoic acid and its receptors. The minimal promoter region of PSG5 gene was activated by RXRalpha but not by RARalpha, in a ligand-dependent manner. The RARE sequence of PSG5 gene promoter was recognized by endogenous RXRalpha present in placental nuclear extracts as well as by RXRalpha either over expressed in cultured non-placental cells or in vitro translated. Mutations at specific nucleotides within the RARE motif abrogated both RXRalpha DNA binding and transcriptional activation of PSG5 promoter mediated by RXRalpha. Moreover, endogenous PSG expression was significantly induced in trophoblast-derived Jeg-3 cells upon 9-cis retinoic acid treatment. Interestingly, the induction level was higher following methotrexate-induced differentiation of Jeg-3 cells to syncytiotrophoblast-like structures. Altogether, these data provide the first evidences demonstrating that transcriptional activity of PSG5 gene is responsive to an external signal involving the retinoids-RXRalpha axis through a conserved RARE motif shared by all PSG gene family members.

High frequency of Panton-Valentine leukocidin genes in invasive methicillin-susceptible Staphylococcus aureus strains and the relationship with methicillin-resistant Staphylococcus aureus in Córdoba, Argentina.

In the study presented here, the genetic characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from patients attending hospitals in the city of Córdoba, Argentina, during 1999-2002 were evaluated to determine their genetic relationship with methicillin-resistant S. aureus (MRSA) clones as part of an effort to control the potential emergence of new epidemic MRSA strains. The results showed there is a high frequency of MSSA strains carrying Panton-Valentine leukocidin genes in invasive infections in Córdoba, Argentina, particularly in those occurring in hospital settings. Panton-Valentine leukocidin genes were found in the genomic background of one clone (ST30-N pulsotype) belonging to a successful internationally distributed MSSA lineage (clonal complex 30), which is closely related to the EMRSA-16 pandemic clone. These genes were also detected in the ancestral clone (ST5-M pulsotype) of the most prevalent MRSA epidemic clone causing healthcare-associated infections in this region, known as the Cordobes/Chilean clone. The molecular characterization of circulating MSSA strains, including the detection of Panton-Valentine leukocidin genes, is thus a useful marker for investigating the evolving epidemiology of hospital- and community-acquired MRSA clones.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina / Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.

Transcriptional control of the human pregnancy-specific glycoprotein 5 gene is dependent on two GT-boxes recognized by the ubiquitous specificity protein 1 (Sp1) transcription factor.

Pregnancy-specific glycoprotein 5 gene (PSG-5) belongs to the human pregnancy-specific glycoprotein family, encoded by eleven highly similar and transcriptionally active genes. High levels of PSG biosynthesis are restricted to the placenta syncytiotrophoblast and are essential for the maintenance of normal gestation in mammalian species. We have investigated here the nature of the transcription factors that recognize the FP1 (-455/-433) and the CPE (-147/-140) regulatory sequences that significantly contribute to basal PSG-5 promoter activity. Both elements bear a similar GT-box motif; and DNA-protein complex formation, as well as promoter activity, is largely dependent on the integrity of these GT-box sequences. Gel shift, super gel shift and UV-crosslinking experiments clearly demonstrate that the ubiquitous specificity protein 1 (Sp1) is the major transcription factor involved in complex formation with both cis-acting elements in normal term placenta tissue and in PSG-non-expressing COS-7 cells. Furthermore, transfection experiments indicate that Sp1 activates PSG-5 promoter constructs. In addition, we show that Sp1 is indeed co-expressed with PSG genes in the syncytiotrophoblast cells, stressing its potential role in the in vivo regulation of PSG expression.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina.

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and "in vivo" studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina.

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and [quot ]in vivo[quot ] studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

Co-localization of KLF6 and KLF4 with pregnancy-specific glycoproteins during human placenta development.

Pregnancy-specific glycoproteins (PSGs) are major placental proteins essential for the maintenance of normal gestation. However, little is known about their gene expression regulation during placentation. It was previously demonstrated that the human core promoter binding protein recently renamed Krüppel-like factor (KLF) 6 binds to a highly conserved sequence within the PSG promoters and is mainly expressed in human term placenta. Here, we determined the expression pattern of the 13 other KLFs during human placental development. We demonstrate that eight KLFs exhibit specific expression patterns in human placental tissues and membranes, in favor of a functional cooperation of specific KLFs during placentation. In addition, we demonstrate that KLF6, KLF4 and PSG proteins are co-expressed in same cell types of placental villi and membranes. This experimental evidence further strengthens the potential cross talk of both transcription factors for PSG gene regulation in vivo.

Aromatase inhibition by an 11,13-dihydroderivative of a sesquiterpene lactone.

Compounds that inhibit aromatase activity are used for the treatment of breast cancer. A group of sesquiterpene lactones inhibit aromatase activity and also exert cytotoxicity through their reactive alpha-methylene-gamma-lactone group. To synthesize sesquiterpene lactones with greater specificity for aromatase inhibition and lower cytotoxicity, we chemically reduced the alpha-methylene-gamma-lactone group in the active aromatase inhibitor 10-epi-8-deoxycumambrin B (compound 1), to obtain the new compound 11betaH,13-dihydro-10-epi-8-deoxycumambrin B (compound 2). Reduction of the alpha-methylene-gamma-lactone group abrogated the cytotoxic activity of compound 1 against the JEG-3, HeLa, and COS-7 cell lines. Compound 2 had higher aromatase inhibitory activity than compound 1 (IC(50) = 2 +/- 0.5 microM versus 7 +/- 0.5 microM, K(i) = 1.5 microM versus 4.0 microM) and was a more potent type II ligand to the heme iron present in the cytochrome P450(arom) active site. Compound 2 inhibited aromatase activity in JEG-3 cells in a comparable manner to the inhibitor aminoglutethimide (AG) used clinically for the treatment of breast cancer. Additionally, compound 2 inhibited androstenedione-induced uterine hypertrophy in sexually immature mice (41% of uterine weight suppression for compound 2 versus 51% for AG). We conclude that the anti-aromatase activity of sesquiterpene lactones does not depend on the presence of the highly reactive alpha-methylene-gamma-lactone group, whereas their cytotoxicity does. These findings may facilitate the development of safer agents for breast cancer therapy.

Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements.

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

Molecular mechanisms implicated in galectin-1-induced apoptosis: activation of the AP-1 transcription factor and downregulation of Bcl-2.

Galectins are emerging as a new class of bioactive molecules with specific immunomodulatory properties. Galectin-1 (Gal-1), a member of this family, has been shown to induce apoptosis of mature T cells and immature thymocytes. To gain insight into the intracellular signals transduced by Gal-1 upon binding to mature T cells, we investigated whether this protein triggered activation of the dimeric AP-1 transcription factor. A marked increase in the binding of nuclear extracts to synthetic oligonucleotides containing the AP-1 consensus sequence, could be detected by an electrophoretic mobility shift assay, when T cells were cultured for 30 min in the presence of Gal-1. This DNA-binding activity was preceded by a rapid increase in the levels of c-Jun mRNA, as determined by Northern blot analysis. Requirement of AP-1 for Gal-1-induced apoptosis was confirmed by the dose-dependent reduction on the level of DNA fragmentation observed when cells were pre-treated with curcumin (an inhibitor of AP-1 activation) before exposure to Gal-1. Finally, evidence is also provided by Western blot analysis, showing that Gal-1 inhibits Concanavalin A (Con A) induction of Bcl-2 protein. Results presented in this study provide the first experimental evidence regarding AP-1 and Bcl-2 as targets of the signal transduction pathway triggered by Gal-1 and set the basis for a more in depth understanding of the molecular mechanisms of T-cell death regulation.

Trypanosoma cruzi-induced immunosuppression: B cells undergo spontaneous apoptosis and lipopolysaccharide (LPS) arrests their proliferation during acute infection.

Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.

The Krüppel-like core promoter binding protein gene is primarily expressed in placenta during mouse development.

The human core promoter binding protein (hCPBP) has been identified as a DNA-binding protein involved in the regulation of TATA box-less genes like those encoding the pregnancy-specific glycoproteins. Structurally, hCPBP contains three zinc fingers in the C-terminal domain, which is highly conserved in a number of proteins that constitute the Krüppel-like family of transcription factors. In the present work, we report the molecular cloning of the mouse CPBP (mCPBP) and its expression pattern during development as well as in adult tissues. The mouse cDNA encodes a protein of 283 amino acids that share 94.4% of identity with the hCPBP. The highest level of mCPBP transcript was detected in placenta, and its expression was lower in total embryos and in adult tissues. We also show by in situ hybridization that during embryonic development the mCPBP gene is mainly expressed in extra-embryonic structures throughout gestation; essentially no specific expression was detected in embryonic tissues. Our data demonstrate that CPBP transcript is enriched in the trophoblastic tissue and strongly suggest that its encoded polypeptide regulates target genes involved in placental development and pregnancy maintenance.

Identification of Trypanosoma cruzi zymodemes by kinetoplast DNA probes.

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.

Differential expression of a tumor necrosis factor receptor-related transcript in gestational trophoblastic diseases in women.

Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the "death domain" region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential "death domain" in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.

A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene.

We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.

In vivo association of ATFa with JNK/SAP kinase activities.

The human ATFa proteins belong to the CREB/ATF family of transcription factors. We have previously shown that the ATFa proteins may contribute to the modulation of the transcriptional activity of the Jun/Fos complexes (Chatton et al. (1994). Oncogene, 9, 375-385). We now show that a protein kinase activity is strongly associated with ATFa in vivo, as revealed by coimmunoprecipitation of ATFa/kinase complexes from whole cell extracts, with antibodies against ATFa. Two independent regions were found to be implicated in kinase binding: a major interaction site is located within the N-terminal 82 residues comprising an important metal-chelating element; a weaker binding site corresponds to the basic sequence element preceding the C-terminal leucine-zipper of ATFa. Induction experiments suggest that each of these ATFa domains may interact with different kinases. The major activity is associated with the ATFa N-terminal domain. Based on its response to various inducers, on both in vitro and in vivo binding assays, and on its immunological properties, this activity most likely corresponds to the 54/55 kDa JNK2 protein. Taken together, these observations suggest that the ATFa proteins, among other CREB/ATF proteins, may be important effectors of cell signalling pathways.

Analyses of cis-acting and trans-acting elements that are crucial to sustain pregnancy-specific glycoprotein gene expression in different cell types.

Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.